Nucleosides consist of a nitrogenous base, either a purine or pyrimidine, and a pentose sugar (ribose). When a phosphate group is present at the 5' position of the ribose nucleosides, the structure is called a nucleotide. Nucleotides form the basic units of nucleic acid, and also serve as intermediates in metabolism, in the form of coenzymes.
The bases characteristic of the deoxyribonucleosides which form deoxyribonucleic acid (hereafter DNA) are thymine, cytosine, adenine and guanine. Those characteristic of the ribonucleosides which comprise ribonucleic acid (hereafter RNA) are uracil, cytosine, adenine and guanosine. Other forms of these nucleosides exist, for example deoxyuridine.
DNA and RNA consist of covalently linked chains of deoxynucleotides or ribonucleotides. The links consist of phosphodiester bridges between the 5'-hydroxyl group of one nucleotide and the 3' hydroxyl group of the next. The nitrogenous bases of the linked nucleotides protrude as side chains in DNA and RNA.
Nucleic acid hybridization probes consisting of sequences of deoxyribonucleotides (DNA) or ribonucleotides (RNA) are well-known the art. Typically, to construct a probe, selected target DNA is obtained as a single strand and copies of a portion of the strand are synthesized in the laboratory and labelled using radioactive isotopes, fluorescing molecules or enzymes that react with a substrate to produce a color change. When exposed to complementary strands of target DNA, for example in a sample of tissue fluid taken from a patient, the labelled DNA probe binds to (hybridizes) its complementary DNA sequence. The label on the probe is then detected and the DNA of interest is thus located. The probe may also be used to target RNA sequences. Finally, probes constructed of RNA sequences may be used to hybridize with a single complementary strand of double-helical DNA forming heteroduplexes without necessitating complete denaturation of the double-helical DNA. Thomas et al., Proc. Nat. Acad. Sci, 73, p. 2294-2298 (1976); Casey and Davidson, Nucl. Acids Res., 4, p. 1539-1552 (1977). DNA probes are proving useful in locating and identifying selected genes, and in the diagnosis and treatment of infection, genetic disorders and cancer. See, U.S. Pat. No. 4,358,535.
A new era in medical sciences has been generated by the remarkable advances made in the field of genetic engineering. The genetic engineering revolution has been hastened by the discovery of naturally occurring enzymes which cleave double helical deoxyribonucleic acid (hereafter DNA) molecules. These enzymes, called restriction endonucleases, cleave DNA molecules at very specific recognition sites within the DNA polymer. These recognition sites are specific sequences of nucleotides for each restriction enzyme. The sequence-specific cleavage of DNA has found many applications such as DNA sequence determinations, chromosome analyses, gene isolation and recombinant DNA manipulations. Other applications include new and useful diagnostic reagents to detect pathogens and aberrant DNA molecules.
The usefulness of restriction endonucleases has been limited to cleavage of DNA molecules containing the nucleic acid sequences recognized by the limited number of these enzymes. In addition, DNA cleavage by restriction endonucleases is limited to the cleavage of DNA at loci where the sequence recognition site occurs. Thus, endonucleases cannot be used to specifically excise a particular piece of DNA unless, by chance, that piece of DNA contains specific nucleic acid sequences recognized by the limited number of known endonucleases.
The development of synthetic reagents for the sequence specific modification of DNA provides additional tools useful in research, diagnostics and chemotherapeutic strategies. For example, the attachment of a DNA-cleaving moiety such as ethylenediaminetetraacetic acid-iron complex, hereinafter EDTA-Fe(II), to a DNA binding molecule produces an efficient DNA cleaving molecule as described by Hertzberg & Devran, J. Am. Chem. Soc. 104, p. 313-315 (1982); Biochemistry 23, p. 3934 (1984). Methidiumpropyl-EDTA (hereinafter MPE), which contains the metal been shown to cleave double helical DNA efficiently in a reaction dependent on ferrous ion (FeII) and dioxygen (O.sub.2). Addition of reducing agents such as dithiothreitol (hereinafter DTT) increases the efficiency of DNA cleavage, as reported by Hertzberg & Dervan, J. Am. Chem. Soc. 104, p. 313-315 (1982); Biochemistry 23 p. 3934 (1984). MPE-Fe(II) cleaves DNA in a relatively non-sequence specific manner and with significantly lower sequence specificity than the enzyme DNase I and is thus useful as a research tool in "footprinting" experiments to identify the binding locations of small molecules such as drugs and proteins on native DNA. Van Dyke & Dervan, Cold Spring Harbor Symp. Quant. Biol. 47, p. 347-353 (1982); Biochemistry 22 p. 2373-2377 (1983); Nucleic Acids Res. 11, p. 5555-5567 (1983); Science 225 p. 1122, (1984).
Many small molecules important in antibiotic, antiviral and antitumor chemotherapy bind to double helical DNA. Until recently knowledge of the DNA base sequence specificities for these small DNA-binding molecules, such as antibiotics, was limited due to the need to rely on the overall binding affinity of such drugs to homopolymer and copolymer DNAS. The attachment of the cleaving complex EDTA-Fe(II) to antibiotics such as distamycin (hereafter DE) followed by DNA cleavage pattern analyses from Maxam-Gilbert sequencing gels has yielded information on the DNA binding sites and orientation of such drugs on DNA. Hertzerg and Dervan, J. Am. Chem. Soc., 104, p. 313-315 (1982); Taylor et al., Tetrahedron, 40, p. 457-465 (1984); Science, 225, p. 1122-1127 (1984).
The mechanism of cleavage by EDTA-FeII complexed with synthetic molecules such as methidium or antibiotics such as distamycin is thought to occur by a common mechanism wherein MPE or DE bind in the minor groove of the right-handed DNA helix by hydrophobic and hydrogen bonding interactions and the cleavage most likely involves diffusible hydroxyl radical. Hertzberg and Dervan, Biochemistry (in press 1984); Tetrahedron, 40, pg. 457-465 (1984).
The above described methods for sequence-specific DNA cleavage have been limited to double-stranded DNA and to those sequences of DNA recognized by antibiotics and DNA intercalators such as methidium. It would provide increased specificity and flexibility with regard to the possible target nucleic acid sequences if sequence-specific cleavage of single stranded nucleic acid and a wider variety of nucleic acid sequences could be accomplished.